Labquality External Quality Assessment Programmes General Bacteriology 1 3/2014

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1 Labquality External Quality Assessment Programmes General Bacteriology 1 3/2014 Photos and text: Markku Koskela, M.D., Ph.D. Clinical microbiology specialist Nordlab Oulu, Finland

2 Specimen 31/2014 Abscess on a toe of an elderly patient Finding: Staphylococcus lugdunensis, significant pathogen. Lab exercises: Isolation and identification of the pathogen. Antimicrobial susceptibility tests.

3 Specimen 31/2014 Abscess on a toe of an elderly patient Growth on sheep blood and chocolated blood agar plates in 5% CO 2, at 35 C, after 24 to 48 hours. -> Middle-sized, yellowish, glistening, beta-haemolytic colonies

4 Specimen 31/2014. (3/2010) Staphylococcus lugdunensis Growth on a sheep blood agar plate in 5% CO 2, at 35 C, after 24 hours. Yellowish, flat, glistening, beta-haemolytic staphylococcal colonies, similar with S aureus colonies. Gram-positive cocci in clusters

5 Specimen 31/2014 (3/2010). Staphylococcus lugdunensis S.lugdunensis gives a positive reaction in staphylococcal agglutination tests like S.aureus, Staphaurex Remel Europe Ltd, UK Pasteurex TM Staph Plus, Bio-Rad, France S.lugdunensis cannot coagulate rabbit plasma like S.aureus ie. its a coagulase negative staphylococcus! Rabbid plasma coagulation: S lugdun negat S aureus posit

6 Specimen 31/2014 (3/2010). Staphylococcus lugdunensis S aureus S lugdunensis Growth on a S aureus identification agar (SAID, biomerieux, France) within 24 hours at +35 C: S aureus green and S lugdunensis graywhite.

7 Specimen 31/2014 (3/2010). Staphylococcus lugdunensis 1. API20Staph: Staphylococcus lugdunensis ( 97 %) - + +, + + -, + - -, - + -, + - -, + - +, Vitek-2 GP Staphylococcus lugdunensis

8 Specimen 32/2014 Maxillary sinus excretion. Sinusitis in a 36-year-old warehouse worker. Findings: Arcanobacterium haemolyticum, a significant / possible pathogen. Streptococcus viridans and Neisseria sicca, normal flora of upper respiratory tract. Lab exercises: Isolation and identification of the bacteria. Interpretation of the findings.

9 Specimen 32/2014. Maxillary sinus excretion. Growth on sheep blood agar plate in 5% CO 2, at 35 C, after 24 to 48 hours.

10 Specimen 32/2014. Maxillary sinus excretion Growth on sheep blood agar plate in 5% CO 2, at 35 C, after 24 to 48 hours. 1. Beta-haemolytic colonies. 2. Alpha-haemolytic colonies. 3. Yellowish, dry, convex, non-haemolytic colonies, which are oxidase positive.

11 Specimen 32/2014. Maxillary sinus excretion Growth on chocolated sheep blood agar plate in 5% CO 2, at 35 C, after 24 to 48 hours. 1. Gray colonies. 2. Green colonies. 3. Yellowish, convex, dry colonies.

12 Specimen 32/2014. Maxillary sinus excretion. 1 2 Growth on streptococcal sheep blood agar plate in 5% CO 2, at 35 C, after 24 to 48 hours. 1. Beta-haemolytic colonies. 2. Alpha-haemolytic colonies.

13 Specimen 32/2014 (2/2011). 1. Arcanobacterium haemolyticum A haemolyt S aureus 1 Catalase- Catalase+ Aerobic growth on streptococcal blood agar plate in 5% CO 2 at +35 C after 72 hours incubation. Beta-haemolytic streptococcal-like colonies that do not agglutinate with beta-haemolytic streptococcal grouping latex reagents. A gram + rod

14 Specimen 32/2014 (2/2011). 1. Arcanobacterium haemolyticum S aureus growth line B-streptoc. growth line A haemol. growth line 1 A beta-haemolytic rousette formation caused by group B beta-haemolytic streptococcus (CAMP test +) Narrowing of the betahaemolytic zone of S aureus caused by Arcanobacterium haemolyticum. (Reverse CAMP test +) CAMP test results on a blood agar plate in 5% CO 2 at +35 C after 24 hours.

15 Specimen 32/2014 (2/2011). 1. Arcanobacterium haemolyticum 1. RapID CB Plus Arcanobacterium haemolyticum 99% , + + -, + + +, - + +,+ - +, Additional tests: catalase -, pigmentation of colonies - ie Vitek-2 ANC Arcanobacterium haemolyticum 97%

16 Specimen 32/2014 (15/2010). 2. Streptococcus viridans Growth on a sheep blood agar plate in 5% CO 2, at 35 C, after 24 hours. An alpha-haemolytic streptococcus which is resistant to optochin.

17 Specimen 32/ Neisseria sicca Oksidaasi+ Growth on a sheep blood agar plate in 5% CO 2, at 35 C, after 24 hours. Yellowish, dry convex colonies. Gramnegat diplokokki

18 Specimen 32/ Neisseria sicca 1. RapID NH 1710 Neisseria sicca/subflava 99,9% + - -, + + +, + - -, Vitek-2 NH Neisseria sicca 95%

19 Specimen 33/2014 Piece of cardiac valve. Subacute endocarditis in a middle-aged male. Finding: Cardiobacterium hominis, significant pathogen. Lab exercises: Isolation and identification of the bacterium. Interpretation of the finding.

20 Specimen 33/2014. Piece of cardiac valve. Gram-stain findings of the specimen: Gram-negative rods

21 Specimen 33/2014. Piece of cardiac valve. Oxidase posit Growth on sheep blood agar plate in 5% CO 2, at 35 C, after 48 hours. Gray, glistening, convex colonies, which are oxidase positive and catalase negative. Catalase negat

22 Specimen 33/2014. Piece of cardiac valve. Growth on a chocolated blood agar plate in 5% CO 2, at 35 C, after 48 hours. Gray, glistening, convex colonies.

23 Specimen 33/2014. Piece of cardiac valve. Primary anaerobic culture Growth on anaerobic blood agar plate (FAA) at 35 C, after 48 hours. Small, gray colonies, which are sensitive to tobramycin and resistant to metronidazole. The same bacterium as on the aerobic plates? ->Aerotolerance test and Gram-stain.

24 Specimen 33/2014 Cardiobacterium hominis Oxidase posit. Growth on chocolated blood agar plate in 5% CO 2, at 35 C, after 48 hours. Convex, yellowish colonies, which are oxidase positive and catalase negative. Pleomorphic Gram-negative rods.

25 Specimen 33/2014 Cardiobacterium hominis 1. RapID NH 2314 Cardiobacterium hominis (99,9 %) - + -, + + -, + - -, Vitek-2 NH Cardiobacterium hominis 63%

26 Specimen 34/2014. Pus from pleural empyema. CT-mediated specimen from right pleura of a 72-year-old male. Findings: Actinomyces meyeri and Streptococcus viridans (S parasanguinis), significant pathogens. Lab exercises: Isolation and identification of the bacteria. Interpretation of the findings.

27 Specimen 34/2014. Pus from pleural empyema. Gram-stain findings of the specimen: Gram-positive cocci and rods.

28 Specimen 34/2014. Pus from pleural empyema. Primary aerobic culture. Growth on sheep blood agar plate in 5% CO 2, at 35 C, within 24 to 48 hours. Light gray, convex, alpha-haemolytic streptococcal colonies.

29 Specimen 34/2014. Pus from pleural empyema. Primary aerobic culture. Growth on sheep blood agar plate in 5% CO 2, at 35 C, within 3 to 4 days. 1. Alpha-haemolytic streptococcal colonies. 2. Very small graywhite colonies!!!

30 Specimen 34/2014. Pus from pleural empyema. Primary aerobic culture. Growth on chocolated sheep blood agar plate in 5% CO 2, at 35 C, within 24 to 48 hours. Alpha-haemolytic streptococcal colonies.

31 Specimen 34/2014. Pus from pleural empyema. Primary aerobic culture. Growth on chocolated sheep blood agar plate in 5% CO 2, at 35 C, within 3 to 4 days. Only alpha-haemolytic streptococcal colonies.???

32 Specimen 34/2014. Pus from pleural empyema. Primary aerobic culture. 3 days 4 days Growth on streptococcal sheep blood agar plate in 5% CO 2, at 35 C, after 3 and 4 days. 1. Alpha-haemolytic streptococcal colonies. 2. Very small graywhite colonies!!!

33 Specimen 34/2014. Pus from pleural empyema. Primary anaerobic culture. Growth on anaerobic blood agar plate (FAA) at 35 C, after 48 hours. The same streptococcal growth as on the aerobic plates. Others?

34 Specimen 34/2014. Pus from pleural empyema. Primary anaerobic culture. Growth on anaerobic blood agar plate (FAA) at 35 C, after 4 days. 1. Streptococcal growth 2. Very small dry colonies. ->Aerotolerance test and Gram-stain.

35 Specimen 34/2014. Pus from pleural empyema. Primary anaerobic culture. 2 days 4 days 1 2 Growth on anaerobic phenyl-ethyl-alcohol agar (PEA) at 35 C after 2 and 4 days. 1. Streptococcal colonies. 2. Very small, dry colonies. ->Aerotolerance test and Gram-stain.

36 Specimen 34/2014 Aerotolerance tests Blood agar: aerobic; chocolated blood agar: 5% CO 2, FAA: anaerobic 1. Left: The bacterium is growing on all three plates. It is aerobic and facultatively anaerobic bacterium. (Streptococcus viridans). 2. Rigth:The bacterium is growing on all three plates but much better in anaerobic than in aerobic conditions. It is not an obligatively anaerobic bacterium. (Actinomyces mayeri).

37 Specimen 34/ Streptococcus viridans Growth on sheep blood agar plate at 35 after 24 hours. Gram-positive cocci in chains Alpha-haemolytic streptococcous which is restistant to optochin.

38 Specimen 34/ Actinomyces meyeri Growth on an anaerobic blood agar plate (FAA) at 35 C, after 4 days.

39 Specimen 34/ Actinomyces meyeri Catalase negative Gram-positive rods Note! A mayeri is catalase negative. It is growing well both on aerobic and anaerobic conditions. Corynebacterium sp (diphteroids) are catalase positive. Most of them are not growing well on anaerobic conditions.

40 Specimen 34/ Actinomyces meyeri Anaerobic identification test (A I): Kanamycin S, vancomycin S, colistin R and metronidazole R. A Gram-positive bacterium.

41 Specimen 34/ Actinomyces meyeri RapID ANA II Actinomyces mayeri (99,9 %) - - -, + + -, - - +, - + +, + + +, + - -

42 Rattoisaa ja Profusion of rowan berries värikästä kevättä! means a little snow in winter?!

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